ISO 23196:2022
(Main)Water quality — Calculation of biological equivalence (BEQ) concentrations
Water quality — Calculation of biological equivalence (BEQ) concentrations
This document specifies the derivation of biological equivalence (BEQ) concentrations for results of in vitro bioassays which are based on measuring effects on a biological process such as enzyme induction or cellular growth. The concept described here can be used for any biological assay after the proof of its applicability. To derive BEQ concentrations, the effect on a biological process caused by a sample – i.e. the activity of the sample – is expressed in terms of a concentration of a reference compound which results in an equivalent effect on the process. The term "sample" used in this document addresses environmental samples as well as defined mixtures and pure compounds used as test item in a bioassay. BEQ concentrations can be derived for environmental water samples, extracts of environmental water samples including tap water or solutions of pure chemicals or mixtures of chemicals.
Qualité de l’eau — Calcul des concentrations en équivalent biologique (BEQ)
Le présent document spécifie la détermination de concentrations en équivalent biologique (BEQ) à partir de résultats de bioessais in vitro qui sont fondés sur la mesure des effets produits sur un processus biologique tel que l’induction d’enzymes ou la croissance cellulaire. Le concept décrit ici peut être utilisé pour n’importe quel bioessai du moment que son applicabilité a été démontrée. Pour déterminer les concentrations BEQ, l’effet produit par un échantillon sur un processus biologique, c’est-à-dire l’activité de l’échantillon, est exprimé sous la forme de la concentration d’un composé de référence à laquelle un effet équivalent est obtenu sur le processus. Le terme «échantillon» utilisé dans le présent document renvoie à des échantillons environnementaux ainsi qu’à des mélanges déterminés et à des composés purs utilisés comme objets de mesure dans le cadre d’un bioessai. Les concentrations en équivalent biologique peuvent être déterminées pour des échantillons d’eau environnementaux, des extraits d’échantillons d’eau environnementaux, y compris d’eau du robinet, ou des solutions de substances chimiques pures ou de mélanges de substances chimiques.
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INTERNATIONAL ISO
STANDARD 23196
First edition
2022-02
Water quality — Calculation of
biological equivalence (BEQ)
concentrations
Reference number
© ISO 2022
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ii
Contents Page
Foreword .iv
1 Scope . 1
2 Normative references . 1
3 Terms and definitions . 1
4 Principle . 2
5 Procedure .5
5.1 General . 5
5.2 Procedure for the calculation of biological equivalence (BEQ) concentrations . 5
5.2.1 Assessment of the suitability of the experimental data for the calculation
of a biological equivalence (BEQ) concentration . 5
5.2.2 Fitting of concentration-effect data for the reference compound . 8
5.2.3 Calculation of quality measures for the fit . 9
5.2.4 Normalization of data from the reference compound and samples . 10
5.2.5 Calculation of the x %-effect level of the reference concentration-effect
relationship and the respective RC -value . 11
x
5.2.6 Assessment of the validity of the experimental data for the calculation of a
biological equivalence (BEQ) concentration .12
5.2.7 Calculation of the concentration factor of the sample at x %-effect level by
linear interpolation .12
5.2.8 Calculation of the biological equivalence (BEQ) concentration .13
6 Validity criteria .14
7 Test report .14
Annex A (informative) Illustration of χ statistics using data from Table 1 .15
Bibliography .18
iii
Foreword
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This document was prepared by Technical Committee ISO/TC 147, Water quality, Subcommittee SC 5,
Biological methods.
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iv
INTERNATIONAL STANDARD ISO 23196:2022(E)
Water quality — Calculation of biological equivalence
(BEQ) concentrations
1 Scope
This document specifies the derivation of biological equivalence (BEQ) concentrations for results of in
vitro bioassays which are based on measuring effects on a biological process such as enzyme induction
or cellular growth. The concept described here can be used for any biological assay after the proof of its
applicability.
To derive BEQ concentrations, the effect on a biological process caused by a sample – i.e. the activity
of the sample – is expressed in terms of a concentration of a reference compound which results in an
equivalent effect on the process. The term "sample" used in this document addresses environmental
samples as well as defined mixtures and pure compounds used as test item in a bioassay. BEQ
concentrations can be derived for environmental water samples, extracts of environmental water
samples including tap water or solutions of pure chemicals or mixtures of chemicals.
2 Normative references
There are no normative references in this document.
3 Terms and definitions
For the purposes of this document, the following terms and definitions apply.
ISO and IEC maintain terminology databases for use in standardization at the following addresses:
— ISO Online browsing platform: available at https:// www .iso .org/ obp
— IEC Electropedia: available at https:// www .electropedia .org/
3.1
biological equivalence concentration
BEQ concentration
concentration of a reference compound (3.6) that causes the same effect as the effect measured in a
sample, a sample dilution or a solution containing one or more chemicals
3.2
concentration-effect relationship
response to a concentration gradient of an environmental sample or a known substance or mixture of
substances which is described by pre-determined diagnostic indicators
[SOURCE: ISO 6107:2021, 3.127, modified — the term "environmental sample" added; Note 1 to entry
has been deleted.]
3.3
concentration factor
CF
ratio of the actual concentration of the sample compared to the original sample taking all enrichment
and dilution steps of the sample into account
3.4
limit of quantification
LOQ
lowest value of a determinant that can be determined with an acceptable level of accuracy and precision
[SOURCE: ISO 15839:2003, 3.18]
3.5
negative control material
well characterized material and/or substance that, when evaluated by a specific test method,
demonstrates the suitability of the test system to yield a reproducible, appropriately negative,
non-reactive or minimal response in the test system
Note 1 to entry: In practice, negative controls include blanks, vehicles/solvents and reference materials.
[SOURCE: ISO 7405:2018, 3.4, modified — Note 1 to entry has been deleted.]
3.6
reference compound
RC
compound with one or more property values that are sufficiently reproducible and well established to
enable the calibration of the measurement method
Note 1 to entry: For the purpose of this document, a reference compound is any well characterized material and/
or substance that, when tested by the procedure described, demonstrates the suitability of the procedure to yield
a reproducible, predictable positive response.
[SOURCE: ISO 7405:2018, 3.5, modified — "compound" replaces "material"; "the calibration of the
measurement method" replaces "use of the material or substance for the calibration of an apparatus,
the assessment of a measurement method or for the assignment of values to materials". Note 1 was
modified to cover only a reference compound resulting in a positive response – otherwise the proposed
concept is not applicable.]
3.7
x %-effect concentration
EC
x
concentration at which a specific effect is detected; x is the percentage (e.g. 10, 25, 50) of this effect, e.g.
growth inhibition
[SOURCE: ISO 15952:2018, 3.6]
3.8
x %-effect concentration of a reference compound
RC
x
concentration of a reference compound (3.6) at which a specific effect is detected; x is the percentage
(e.g. 10, 25, 50) of this effect, e.g. growth inhibition
4 Principle
The general idea for the derivation of BEQ concentrations, assessed by the test method, is shown in
Figure 1. The biological activity of a sample is expressed in terms of a concentration of a reference
compound which affects a biological process to the same extent. By this means, BEQ concentrations
allow an indirect quantification of results and a comparison of results obtained by different laboratories.
Furthermore, a robust way to calculate BEQ concentrations is a necessary requirement for a possible
[5],[6],[7],[8]
use of effect-based trigger (EBT) values in regulations .
Key
X concentration of the reference compound, e.g. in ng/l
X1 concentration factor of the sample compared to the original water sample
Y effect in arbitrary units
1 sample diluted
2 sample concentrated
3 biological equivalence (BEQ) concentration
reference compound
sample
NOTE The measured effect of a sample or a sample dilution is extrapolated to a concentration of a reference
compound which induces the bioassay to the same extend as the sample or sample dilution to derive a biological
equivalence (BEQ) concentration, here an example of an agonistic action is shown.
Figure 1 — Basic principle of the derivation of a biological equivalence (BEQ) concentration
Several different methods for the derivation of BEQ concentrations have been published, but not all
methods are applicable to all kinds of concentration-effect relationships. For a detailed discussion about
advantages and disadvantages of the various mathematical approaches, see References [1],[3],[5],[6].
The method described in this document, termed RC -approach, is reported to be a robust method for
x
[11],[12] [13]
the derivation of BEQ concentrations and outlined as well in OECD 455 .
NOTE In the literature, other terminologies might be found for RC , such as PC . PC describes the
x 10 10
concentration of the positive control that induces a 10 % effect-level. In this document, the term “reference
compound” is used instead of “positive control”. Therefore, RC is used instead of PC.
The approach is illustrated in Figure 2. As a first step of the RC -approach, the concentration-effect
x
relationship of the reference compound is modelled to determine the maximum effect of the assay for
the reference compound. Next, the effect levels are normalized to a percentage scale whereby the effect
level of the negative control material is defined as 0 % and the maximum effect level of the reference
compound is defined as 100 %. Then, the concentration of the reference compound that affects the
assay to the x %-effect level is calculated (RC ).
x
Key
X concentration of the reference compound, e.g. in ng/l
X1 concentration factor of the sample compared to the original water sample
Y normalized effect in %
RC concentration of the reference compound at 10 % effect level
CF concentration factor of the sample at 10 % effect level with respect to the reference compound
reference compound
sample
NOTE Following data normalization, where 0 % equals the effect in the negative control material and 100 %
is the modelled maximum effect of the reference. The sample concentration factor (CF) and reference compound
concentration (RC) required to rea
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